Carbopyronine fluorescent dyes

ABSTRACT

The invention relates to the use of carbopyronine compounds of general formula (I) as marker groups in methods for detecting analytes. The invention also relates to novel carbopyronine compounds and to a method for producing same.

The invention relates to the use of carbopyronine compounds of thegeneral formula (I) as labeling groups in procedures for the detectionof analytes, to novel carbopyronine compounds and to a process for thepreparation of these compounds.

In chemical, medical and biological analysis, dyes are used as labelingor detection groups. In particular, fluorescent dyes have gainedimportance in recent years and displaced other often cost-intensiveprocedures, which use, for example, radioisotopes for labeling.

In particular in the field of DNA sequencing, fluorometric procedureshave gained acceptance in recent years and almost completely replacedthe procedures customary up till then, which use radioactive isotopes.

In spite of the availability of various fluorescent dyes, such as, forexample, FITC (fluorescein isothiocyanate), FLUOS (fluoresceinN-hydroxysuccinimide ester), rhodamine derivatives etc., it waspreviously not possible to solve the problems due to backgroundfluorescence, honspecific binding phenomena and the need forcost-intensive measuring equipment in a satisfactory manner.

As a result of background fluorescence and nonspecific binding, thesensitivity and accuracy of the measurements is reduced. In addition, inthe case of available fluorescent dyes the absorption maximum lies inregions which do not make possible the use of light sources which areless expensive and which can be of small dimensions, such as, forexample, He/Ne lasers and laser diodes.

An object of the present invention was thus to make availablefluorescent dyes which can be employed as labeling groups in proceduresfor the detection of analytes and at least partially avoid thedisadvantages of the prior art.

This object has been achieved by the use of compounds of the generalformula (I)

as labeling groups in a procedure for the detection of an analyte, where

R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are in each case independently hydrogen,halogen, a hydroxyl, amino, sulfo or carboxyl or aldehyde group or asaturated or unsaturated, straight-chain, branched or cyclic hydrocarbongroup having up to 20 C atoms, where the hydrocarbon groups includealkyl, alkenyl, alkynyl, cycloalkyl, aryl, in particular phenyl, or/andheteroaryl radicals and optionally heteroatoms such as oxygen, sulfur ornitrogen atoms or/and two or more substituents, preferably selected fromhalogens, hydroxyl, amino, sulfo, phospho, carboxyl, aldehyde,C₁-C₄-alkoxy or/and C₁-C₄-alkoxycarbonyl groups, or one or more of theradicals R₁-R₇, in each case with adjacent substituents, form a ringsystem which can contain one or more multiple bonds,

R₈ and R_(8a) in each case independently are a saturated or unsaturated,straight-chain, branched or cyclic hydrocarbon group having up to 20carbon atoms, e.g. a C₁-C₆-alkyl group, in particular methyl, ethyl,propyl or/and butyl, or an aryl or heteroaryl group, in particularphenyl, which optionally contain heteroatoms such as oxygen, sulfur ornitrogen atoms or/and one or more substituents, preferably selected fromhalogens, hydroxyl, amino, sulfo, phospho, carboxyl, aldehyde,C₁-C₄-alkoxy or/and C₁-C₄-alkoxycarbonyl groups, or R₈ and R_(8a) canform a ring system,

R₉, R₁₀, R₁₁ and R₁₂ in each case independently are hydrogen or asaturated or unsaturated, straight-chain, branched or cyclic hydrocarbongroup having up to 20 C atoms, e.g. polyether, phenyl, phenylalkylhaving 1-3 C atoms in the chain, where the hydrocarbon groups canoptionally contain heteroatoms such as oxygen, sulfur or nitrogen atomsor/and one or more substituents, preferably selected from halogens,hydroxyl, amino, sulfo, phospho, carboxyl, carbonyl, alkoxy or/andalkoxycarbonyl groups,

or one or more of the radicals R₉-R₁₂, in each case with adjacentsubstituents, form a ring system which can contain one or more multiplebonds,

where —N(R₁₁)(R₁₂) or/and ═(R₉)(R₁₀) can be replaced by —OR⁹ or/and ═O,

and X is optionally anions present for charge equalization.

The compounds of the general formula (I) can be employed as labelinggroups in procedures for the qualitative or/and quantitativedetermination of an analyte. This determination can be carried out inaqueous liquids, e.g. samples of body fluids such as, for example,blood, serum, plasma or urine, wastewater samples or foodstuffs. Theprocedure can also be carried out as a wet test, e.g. in a cuvette, oras a dry test in an appropriate reagent carrier. The determination ofthe analyte can be carried out here by means of a single reaction or bymeans of a sequence of reactions. Surprisingly, the use of compounds ofthe general formula (I) showed very good results in chemical and inparticular in medical and biological detection procedures for thedetermination of an analyte, especially in nucleic acid sequencingprocedures and in protein analysis.

The compounds of the general formula (I) can be used in all chemical,medical and biological detection procedures known to the person skilledin the art in which fluorescent dyes are suitable as labeling groups.For this, the compounds of the general formula (I) are in generalcoupled covalently to a receptor which is specific for the analyte to bedetected. This takes place using generally known procedures. Thespecific receptor can be any suitable compound or any suitable molecule,preferably it is a peptide, a polypeptide or a nucleic acid. Thecompounds or conjugates of these compounds can be used, for example, innucleic acid, hybridization procedures, in particular for the sequencingof nucleic acids or immunochemical procedures. Procedures of this typeare described, for example, in Sambrook et al., Molecular Cloning, ALaboratory Manual, 1989, Cold Spring Harbor.

A further object of the present invention was to make available novelcarbopyronine compounds which are suitable in particular for use aslabeling groups in analyte detection procedures, can be prepared usingsimple and inexpensive processes, can be handled without problems and atleast partially avoid the disadvantages of the prior art.

This object has been achieved by a compound of the general formula (I)

where

R₁-R₁₂ and X have the meanings indicated above,

with the proviso that if R₁-R₃ and R₅-R₇ are hydrogen and R₈, R_(8a) andR₉-R₁₂ are methyl,

R₄ is not hydrogen, methyl, isopropyl, phenyl, 2,6-dimethylphenyl or2-isopropenylphenyl.

An advantage of the compounds (I) is that owing to an almost arbitrarysubstituent variation the properties of individual compounds, e.g. thespectroscopic properties, the position of the absorption andfluorescence maxima, the solubility properties, the fluorescence quantumyield and decay time, vary strongly and thus can be selected as desired.In this way, interferences with interfering substances in samples suchas serum, blood or plasma etc. can be reduced or even avoidedcompletely. The preparation of some compounds of the formula (I) can becarried out by processes known per se. Preferably, the synthesis iscarried out, however, according to a novel process described below,which is particularly simple and inexpensive.

In a preferred class of the compounds (I), R₆ is bridged with R₁₁ or/andR₇ with R₁₂, R₁ with R₁₀ or/and R₂ with R₉ and form a ring system whichcan contain one or more multiple bonds. The ring system preferablycontains one or more 5- or 6-membered rings.

R₄ is preferably hydrogen, C₁-C₆-alkyl or a radical containing anaromatic ring system, e.g. a radical containing a carboxyl or/andhalogen group, such as 2-carboxyphenyl, 2-carboxytetrachlorophenyl orpentafluorophenyl. R₈ and R_(8a) are preferably in each caseindependently methyl, ethyl or/and optionally substituted phenyl.

Examples of particularly preferred classes of compound are shown in thegeneral formulae IVa to IVe:

in which the dashed lines are optionally double bonds, in whose presencethe radicals R bonded via a dashed line are absent,

R₁, R₃, R₄, R₅, R₆, R₇, R₈, R_(8a), R₉, R₁₁, R₁₂ and X are as definedabove, and R, on each occurrence, can be identical or different and isdefined as R₁-R₇ above.

The compounds preferably have a group capable of covalent coupling, e.g.—COOH, —NH₂, —OH or/and —SH. By means of this coupling group, thecompound can be coupled to a carrier or/and to a biomolecule accordingto known methods. The carrier can consist of any material which issuitable, in particular for detection procedures, e.g. of porous glass,plastics, ion-exchange resins, dextrans, cellulose, cellulosederivatives or/and hydrophilic polymers. The biomolecules are preferablyselected from peptides, polypeptides, nucleotides, nucleosides, nucleicacids, nucleic acid analogs or/and haptens.

Surprisingly, the absorption maxima and the fluorescence quantum yieldare not significantly changed by coupling of the compounds according tothe invention to the abovementioned carriers and biomolecules.

Actual examples of compounds according to the invention are shown intable 1 below.

TABLE 1 λ_(A): absorption maximum λ_(F): fluorescence maximum Q_(F):fluorescence quantum yield in ethanol Structure λ_(A)/nm λ_(F)/nmQ_(F)/% 1 Cp 149

606 627 71 2 AZ 6

608 630 65 3 JA 261

608 630 70 4 JA 262

608 630 70 5 AZ 1

617 641 77 6 AZ 4

617 641 78 7 AZ 14

617 641 78 8 AZ 7

618 642 75 9 JA 260

616 640 75 10 JA 264

616 640 75 11 JA 263

616 640 76 12 JA 266

616 640 76 13 JA 265

634 658 62 14 AZ 8

641 666 60 15 JA 267

633 660 60 16 JA 268

634 660 58 17 AZ 2

633 657 63 18 AZ 5

633 657 61 19 AZ 3

629 650 69 20 AZ 13

626 648 87 21 AZ 9

647 675 55 22 AZ 12

647 664 58 23 AZ 11

664 688 49 24 JF 19

602 643 58 25 JF 20

604 675 41 26 JF 18

601 636 67 27 JF 16

611 638 6 28 JF 21

610 637 46 29 JF 22

612 641 41 30 JF 24

617 643 71 31 JF 25

613 638 6 32 JF 26

611 640 59 33 JF 17

610 640 70 34 JF 23

618 643 60 35 AZ 16

606 628 70 36 AZ 17

615 640 75 37 AZ 18

627 655 62 38 JF30

621 652 4 39 JF 31

618 648 5 40 JF 32

618 647 5 41 JF 34

612 642 75 42 JF 35

642 672 64 43 JF 36

632 662 85 44 JF 37

662 692 60 45 JF 38

653 683 70 46 JF 39

683 713 45 47 JF 40

670 700 55 48 JF 41

700 730 40 49 JF 42

557 577 95 50 JF 43

632 660 80

A further object of the present invention consisted in making availablea preparation process for carbopyronine compounds which can be carriedout in a simple, environmentally compatible and inexpensive manner andwhich at least partially avoids the disadvantages of the known processesfor the preparation of carbopyronines.

This object was achieved according to the invention by a process for thepreparation of compounds of the general formula (I)

where R₁-R₁₂ and X have the meanings indicated in claim 1, characterizedin that a compound of the general formula (II)

in which R₅, R₆, R₇, R₈, R_(8a), R₁₁ and R₁₂ are as defined above, orthe dehydration product of II is reacted with a compound of the generalformula III

in which R₁-R₄, R₉ and R₁₀ are as defined above and Y is a halogen, inparticular bromine, a hydroxyl or thio group, in a suitable solvent,under acidic conditions and in the presence of a catalyst and thecompound formed by ring closure between the compounds II or theirdehydration product and III is reacted by oxidation to give thestructure I.

In the process, it is possible to use all suitable solvents which arecompatible with the starting materials, the products and the catalyst,preferably boron trichloride. The solvent is preferably a nonpolarsolvent, in particular methylene chloride, 1,2-dichloroethane orchloroform.

The acids employed can be customary acids. The acid is preferably aninorganic acid such as sulfuric acid, phosphoric acid or polyphosphoricacid.

The oxidants used can likewise be customary oxidants. The oxidanttetrabutylammonium(meta)periodate is preferred.

It is particularly advantageous that the process can be carried outwithout isolation of intermediates. This leads to a reduction in theexpenditure of time, labor and material.

The invention is illustrated in greater detail by the examples below.The FIGS. 1, 2, 3 and 4 show the absorption and fluorescence spectra ofthe compounds according to the invention AZ 2 (17), AZ 13 (20), JA 268(16) and AZ11 (23).

EXAMPLES

A. Preparation Process According to the Invention for CarbopyronineCompounds

In the process according to the invention,4-N,N-dimethylaminobenzylsulfanilic acid, which is used in the processaccording to Aaron and Barker (J. Chem. Soc. (1963), 2655) is replacedby 4-hydroxymethyl-N,N-dimethylaniline and reacted with the isopropenylderivative to give the carbopyronine in the presence of borontrichloride solution as a catalyst. The reaction mixture can be reactedwith concentrated sulfuric acid to give the leuko base of the dyewithout isolation of the intermediate. The oxidant lead dioxide used inAaron and Barker (loc. cit.) is replaced bytetrabutylammonium(meta)periodate. To this end, the ethanolic solutionof oxidant and leuko base is heated to boiling, it being possible todetect by thin-layer chromatography that the oxidation is alreadycomplete after a few minutes.

After the oxidation, the carbopyronine is precipitated from ethanolicsolution as a poorly soluble perchlorate by addition of 10% strengthsodium perchlorate solution and slow dropwise addition of water.

The novel synthesis route can be employed universally. The correspondingalcohols can be obtained from aniline, indoline, tetrahydroquinoline and1,2-dihydroquinoline derivatives by a Vilsmaier synthesis withsubsequent reduction and these can be reacted with an isopropenylderivative to give the dye. Unlike the synthesis of Aaron and Barker,the synthesis proceeds in one step, i.e. isolation of intermediates isnot necessary.

The synthesis procedures for the compounds JA 261, JA 262, AZ 4, AZ 14,JA 267, JA 268, JF 19, JF 22 and JF 17 are presented below.

B. Synthesis Examples

Compound JA 261

1 g (4 mmol) of ethyl N-methyl-N-(4-hydroxymethylphenyl)-4-aminobutyrateand 0.71 g (4.4 mmol) of 3-(isopropenyl)-N,N-dimethylaniline aredissolved in 20 ml of methylene chloride. 4 ml of a 1 molar BCl₃solution (in methylene chloride) are slowly added with stirring and icecooling. The solution is stirred overnight at room temperature. Thereaction mixture is then added dropwise to 20 g of concentrated sulfuricacid, which is cooled in an ice/methanol bath. The mixture is stirreduntil a homogeneous solution is present. The methylene chloride isdistilled off on a rotary evaporator. The sulfuric acid solution isstored overnight in a refrigerator. The solution is then poured onto iceand neutralized with dilute sodium hydroxide solution. The aqueoussolution is extracted with chloroform. The combined organic phases aredried over sodium sulfate, filtered and concentrated to dryness on arotary evaporator. The residue is taken up in 200 ml of ethanol andtreated with 10 drops of 60% strength perchloric acid and 0.17 g (0.39mmol) of tetrabutylammonium(meta)periodate. The solution is heated toreflux for 30 min. The cooled solution is added dropwise to a solutionof 20 g of sodium perchlorate in 1 l of water. The mixture is stirredovernight. The green, lustrous precipitate is filtered off and driedover phosphorus pentoxide in a desiccator.

Yield: 0.56 g

¹H-NMR data in CDCl₃:

δ 1.25 (T, 3H, —CH₃); 1.7 (S, 6H, —CH₃); 2.0 (QI, 2H, —CH₂—); 2.5 (T,2H, —CH₂—); 3.3 (S, 9H, N—CH₃); 3.7 (T, 2, —CH₂—); 4.15 (Q, 2H, N—CH₂—);6.85 (DvD, 2H, ArH); 7.05 (D, 1H, ArH); 7.2 (D, 1H, ArH); 7.65 (D, 2H,Ar—H); 8.0 (S, 1H, —CH═)

Compound JA 262

100 mg of JA 261 are dissolved in a mixture of 20 ml of acetone, 40 mlof water and 2 ml of 2 N hydrochloric acid. The solution is heated toreflux (internal temperature: 64° C.). After 24 h, the solution iscooled and treated with 100 ml of 10% strength aqueous sodiumperchlorate solution. The precipitate is filtered off and dried.

Yield: 0.04 g.

Compound AZ 4

1.00 g (4.25 mmol) of ethyl 4-(5-hydroxymethylindolin-1-yl)butyrate and0.76 g (4.25 mmol) of 3-(isopropenyl)-N,N-dimethylaniline are dissolvedin 15 ml of methylene chloride and treated dropwise with 4.25 ml (4.25mmol) of a 1 molar solution of boron trichloride in hexane with icecooling. The reaction mixture is stirred at room temperature for 30 min.The reaction mixture is then added dropwise to 10 ml of concentratedsulfuric acid and stirred at room temperature for 1 h. The deepred-colored reaction mixture is added dropwise to 100 ml of ice-coldethanol, treated with 0.78 g (1.8 mmol) oftetrabutylammonium(meta)periodate and heated to boiling for 3 min. It isallowed to cool to room temperature and is treated with 50 ml of 20%strength sodium perchlorate solution. 300 ml of water are then addeddropwise to precipitate the dye completely. The crystalline product isfiltered off and dried in vacuo in a desiccator using SICAPENT®.

Yield: 0.7 g

¹H-NMR data in acetone-d₆:

δ 0.9 (T, 3H, —CH₃ a); 1.7 (S, 6H, —CH₃ m); 2.47 (T, 2H, —CH₂-c); 3.22(T, 2H, —CH₂— g); 3.34 (S, 6H, N—CH₃ o); 3.8 (T, 2, —CH₂— e); 4.09 (T,2H, —CH₂— f); 4.42 (Q, 2H, —CH₂— b); 6.95 (DvD, 1H, ArH k); 7.22 (D, 1H,ArH 1); 7.3 (S, 1H, ArH n); 7.7 (D, 1H, Ar—H j); 8.08 (S, 1H, —CH=i)

Compound AZ 14

4 g (8 mmol) of AZ 4 are dissolved in 30 ml of water and 20 ml ofacetone and treated with 1 ml of 2 N hydrochloric acid. The reactionmixture is heated to reflux for 18 h. It is treated with 50 ml ofchloroform and the organic phase is separated off. After extraction withchloroform a further three times, the combined organic phases are washedwith water and dried over sodium sulfate. The dye solution isconcentrated to dryness on a rotary evaporator and then purified bycolumn chromatography.

¹H-NMR data in acetone-d₆:

δ 1.72 (S, 6H, —CH₃ k); 2.0 (M, 2H, —CH₂— b); 2.49 (T, 3H, —CH₂— a);3.25 (T, 2H, —CH₂— e); 3.34 (S, 6H, —CH₃ m); 3.81 (T, 2, —CH₂— c); 4.11(T, 2H, —CH₂— d); 6.95 (DvD, 1H, ArH i); 7.22 (D, 1H, ArH j); 7.3 (S,1H, ArH 1); 7.42 (S, 1H, Ar—H f); 7.7 (D, 1H, ArH h); 8.1 (S, 1H, —CH=g)

Compound JA 267

1.2 g (3.8 mmol) of ethyl4-(6-hydroxymethyl-2,2,4-tri-methyl-1,2-dihydroquinol-1-yl)butyrate and0.68 g (3.8 mmol) of 3-(isopropenyl)-N,N-dimethylaniline are dissolvedin 30 ml of methylene chloride. 4 ml of a 1 molar BCl₃ solution inmethylene chloride are added slowly with stirring and ice cooling. Thesolution is stirred at room temperature for 20 min. The reaction mixtureis then added dropwise to 20 ml of conc. sulfuric acid. It is stirreduntil a homogeneous solution is present. The methylene chloride isdistilled off on a rotary evaporator and the sulfuric acid solution isstirred at room temperature for 1 h. The residue is taken up in 400 mlof ice-cooled ethanol. 1.2 g (2.7 mmol) oftetrabutylammonium(meta)periodate are added thereto. The solution isbriefly heated to boiling, cooled and treated with 200 ml of a 20%strength sodium perchlorate solution. 500 ml of water are then addeddropwise. The precipitate is filtered off and dried in a desiccator.

Compound JA 268

1.8 g of JA 267 are heated to reflux for 6 h in a mixture of 50 ml ofacetone, 50 ml of water and 5 ml of 2 N hydrochloric acid. The solventis distilled off and the residue is purified by chromatography.

Compound JF 19

0.27 ml (0.81 mmol) of a 3 M methylmagnesium bromide solution in diethylether are added dropwise within an argon protective gas atmosphere atroom temperature to a solution of 50 mg (0.16 mmol) of2,10-bis(dimethylamino)anthrone in 10 ml of dry tetrahydrofuran. Afterreaction is complete, the reaction mixture is cooled in an ice-waterbath, dissolved in 50 ml of ethanol and acidified with trifluoroaceticacid. This solution is suspended in a mixture of 50 ml of chloroform and50 ml of water. The organic phase is separated off, concentrated todryness on a rotary evaporator and dissolved in ethanol. The solution isthen added dropwise to 100 ml of aqueous 25% strength sodium perchloratesolution. After addition is complete, a further 300 ml of water areadded dropwise. The dye precipitated is filtered and dried in vacuo.

Yield: 0.04 g

Compound JF 22

Under protection by argon, 11 mg (1.6 mmol) of lithium powder (0.5%sodium, Metallgesellschaft) are suspended in 2 ml of dry diethyl ether.A solution of 0.17 g (0.8 mmol) of 1-bromo-2,6-diethylbenzene in 4 ml ofdiethyl ether is added dropwise to this suspension with stirring. Afteraddition is complete, the mixture is stirred at room temperature for 15min. The suspension is filtered through glass wool in order to removethe remaining residues of lithium. The solution thus obtained is addeddropwise at room temperature to a solution of 50 mg (0.16 mmol) of2,10-bis(dimethylamino)anthrone in 10 ml of dry tetrahydrofuran. Afterreaction is complete, the reaction mixture is cooled in an ice-waterbath, dissolved in 50 ml of ethanol and acidified with trifluoroaceticacid. This solution is suspended in a mixture of 50 ml of chloroform and50 ml of water. The organic phase is separated off, concentrated todryness on a rotary evaporator and purified by column chromatography onsilica gel. After the dye fraction has been concentrated to dryness on arotary evaporator, it is dissolved in ethanol and then added dropwise to100 ml of aqueous 25% strength sodium perchlorate solution. A further300 ml of water are then added dropwise. The dye precipitated isfiltered and dried in vacuo.

Yield: 0.02 g

Compound JF 17

0.14 g (0.55 mol) of 2-(2-bromophenyl)-4,4-dimethyl-2-oxazoline isdissolved in 7.5 ml of tetrahydrofuran under protective gas (argon) andcooled to −78° C. 0.7 ml (1.1 mmol) of a 1.6 M solution oft-butyllithium in hexane are added dropwise to this solution such thatthe temperature remains below −75° C. After addition is complete, thesolution is stirred for 15 min. 34 mg (0.11 mmol) of2,10-bis(dimethylamino)anthrone in 2 ml of dry tetrahydrofuran are addedto this solution. The temperature should not exceed −70° C. in thecourse of this. The mixture is then warmed to −60° C. and stirred for 3h. The cooling bath is removed and the mixture is allowed to warm toroom temperature. After 24 h, the reaction mixture is cooled in anice-water bath,. dissolved in 50 ml of ethanol and acidified withtrifluoroacetic acid. This solution is suspended in a mixture of 50 mlof chloroform and 50 ml of water. The organic phase is separated off,concentrated to dryness on a rotary evaporator and purified by columnchromatography. The dye fraction is concentrated to dryness on a rotaryevaporator, taken up in ethanol and subsequently added dropwise to 100ml of aqueous 25% strength sodium perchlorate solution. After additionis complete, a further 300 ml of water are added dropwise. The dyeprecipitated is filtered and dried in vacuo.

Compound AZ 18

1st Stage

3-(N,N-Dimethylamino)triphenylcarbinol

2.8 g (0.12 mol) of magnesium and 10 ml of diethyl ether (absolute) aretreated with 2.6 g (0.02 mol) of broinobenzene. In order to start thereaction, the mixture is slightly warmed. The start of the reaction canbe detected by the turbidity of the reaction mixture. 16.2 g (0.1 mol)of bromobenzene are then dissolved in 15 ml of ether and added dropwiseto the reaction mixture. It is heated to reflux for 1 h, the magnesiumalmost completely dissolving. After cooling in an ice bath, a solutionof 10 g (0.055 mol) of methyl 3-dimethylaminobenzoate in 15 ml ofabsolute ether is added dropwise. After the addition, the reactionmixture is heated to reflux for 2 h, cooled and hydrolyzed dropwise withwater. 50 ml of water and 50 ml of ether are added and the mixture istreated with saturated ammonium chloride solution until the whiteprecipitate has dissolved again. The aqueous phase is extracted withether. The combined organic phases are washed with saturated sodiumhydrogencarbonate solution and with water. The solution is then driedover sodium sulfate and the solvent is distilled off. The residual paleyellow oil can be used directly for the subsequent reaction.

2nd Stage

AZ 18

0.6 g (3 mmol) of N,N-dimethyl-4-hydroxymethylaniline and 0.9 g (3 mmol)of 3-(N,N-dimethylamino)triphenylcarbinol are dissolved in 30 ml ofmethylene chloride. 4 ml of a 1 molar BCl₃ solution in methylenechloride are slowly added with stirring and ice cooling. The solution isstirred at room temperature for 2 h. The reaction mixture is then addeddropwise to 20 ml of 70% strength sulfuric acid. The methylene chlorideis distilled off on a rotary evaporator and the sulfuric acid solutionis stirred at room temperature for 20 h. The residue is slowly dissolvedin 100 ml of ice-cooled ethanol. 1.2 g (2.7 mmol) oftetrabutylammonium(meta)periodate are added thereto. The solution isbriefly heated to boiling, cooled and treated with 100 ml of a 20%strength sodium perchlorate solution. 250 ml of water are then addeddropwise. The precipitate is filtered off and dried in a desiccator.

Compound JF 30

1.85 ml (3.05 mmol) of a 15% strength t-butyllithium solution (inn-pentane) are added at −78° C. to a solution of 0.39 g (1.53 mmol) of2-(4-bromophenyl)-4,4-dimethyl-2-oxazoline in 20 ml of drytetrahydrofuran such that the temperature remains below −70° C. Aftercomplete addition, 150 mg (0.48 mmol) of 3,6-bis(dimethylamino)anthronein 30 ml of dry tetrahydrofuran are added such that the temperatureremains below −60° C. The solution is allowed to warm to roomtemperature and is stirred at room temperature for 18 h. The reactionmixture is cooled in an ice-water bath, dissolved in 50 ml of ethanoland acidified with trifluoroacetic acid. This solution is suspended in amixture of 50 ml of chloroform and 50 ml of water. The organic phase isseparated off, concentrated to dryness on a rotary evaporator, andpurified by column chromatography on silica gel. The dye is eluted using15% strength ethanolic chloroform. After the product phase has beenconcentrated to dryness on a rotary evaporator, it is dissolved inethanol and then added, dropwise to 100 ml of aqueous 25% strengthsodium perchlorate solution. After addition is complete, a further 300ml of water are added dropwise. The dye precipitated is filtered anddried over phosphorus pentoxide in a vacuum desiccator.

Yield: 50% (cryst. substance after chromatography)

Compound JF 31

80 mg (0.14 mmol) of JF 30 are heated under reflux for 40 min in 10 mlof a 1:3 mixture of 2 M hydrochloric acid and acetone. The mixture isallowed to cool and is suspended in 50 ml of a 1:1 mixture of chloroformand water. The water phase is neutralized with saturated sodiumhydrogencarbonate solution. The organic phase is separated off and theaqueous is extracted a number of times with 20% strength ethanolicchloroform. The combined organic phases are concentrated on a rotaryevaporator and purified by column chromatography on silica gel. The dyeis eluted using 20% strength ethanolic chloroform. After the productphase has been concentrated to dryness on a rotary evaporator, it isdissolved in ethanol and then added dropwise to 100 ml of aqueous 25%strength sodium perchlorate solution. After addition is complete, afurther 300 ml of water are added dropwise. The dye precipitated isfiltered and dried over phosphorus pentoxide in a vacuum desiccator.

Yield: 72% (cryst. substance after chromatography)

Compound JF 32

70 mg (0.12 mmol) of JF 31 are heated to reflux for 1 h in a 10%strength sodium hydroxide solution in 1:1 ethanol and water. The mixtureis allowed to cool and is suspended in a 1:1 mixture of chloroform andwater. It is adjusted to pH=8 using trifluoroacetic acid and the organicphase is separated off. The aqueous phase is extracted a number of timeswith 20% strength ethanolic chloroform. This extraction is repeateduntil there is barely still dye in the aqueous phase (testing by meansof acidification). The combined organic phases, are adjusted to pH=2using trifluoroacetic acid, concentrated on a rotary evaporator andpurified by column chromatography on silica gel. The dye is eluted using10% strength ethanolic chloroform. After the product phase has beenconcentrated to dryness on a rotary evaporator, it is dissolved inethanol and then added dropwise to 100 ml of aqueous 25% strength sodiumperchlorate solution. After addition is complete, a further 300 ml ofwater are added dropwise. The dye precipitated is filtered and driedover phosphorus pentoxide in a vacuum desiccator.

Yield: 57% (cryst. substance after chromatography)

Compound JF 42

70 mg (0.12 mmol) of JF 17 are heated to reflux for 1 h in 30 ml of asolution of 3 g of sodium hydroxide in ethanol/water (1:1). The solutionis allowed to cool and is neutralized using semiconcentratedhydrochloric acid. The dye is then precipitated by dropwise addition ofwater. The product is filtered off and dried in a vacuum desiccator overphosphorus pentoxide.

Compound JF 36

25.3 g (0.1 mol) of6-(2-carboxybenzoyl)-N-ethyl-1,2,3,4-tetrahydroquinoline and 20.1 (0.1mol) of N-ethyl-7-isopropenyl-1,2,3,4-tetrahydroquinoline are dissolvedin 500 ml of dichloromethane and treated with 60 g of phosphoruspentoxide. The mixture is heated under reflux for 2 h, allowed to cooland the solvent is distilled off in vacuo. The residue is treated withconc. sulfuric acid. This solution is stirred at room temperature for 30min. After this, the sulfuric acid solution is added to 1 000 ml ofice-cooled ethanol and treated dropwise with 50 ml of 60% strengthperchloric acid and 5 l. The dye precipitated is filtered off and driedover phosphorus pentoxide in a vacuum desiccator.

Compound JF 37

39.1 g (0.1 mol) of6-(2-carboxy-3,4,5,6-tetrachlorobenzoyl)-N-ethyl-1,2,3,4-tetrahydroquinolineand 20.1 (0.1 mol) of N-ethyl-7-isopropenyl-1,2,3,4-tetrahydroquinolineare dissolved in 500 ml of dichloromethane and treated with 60 g ofphosphorus pentoxide. The mixture is heated under reflux for 2 h,allowed to cool and the solvent is distilled off in vacuo. The residueis treated with conc. sulfuric acid. This solution is stirred at roomtemperature for 30 min. After this, the sulfuric acid solution is addedto 1 000 ml of ice-cooled ethanol and treated dropwise with 50 ml of 60%strength perchloric acid and 5 l. The dye precipitated is filtered offand dried over phosphorus pentoxide in a vacuum desiccator.

C. Examples of Conjugate Formation

JA 262 Active Ester

0.1 mmol of JA 262 is dissolved in 20 ml of acetonitrile with 0.2 mmolof N-hydroxysuccinimide and 0.2 mmol of dicyclohexylcarbodiimide. Thesolution is stirred at room temperature for 4 h and the product mixtureis concentrated on a rotary evaporator. Purification is carried out bychromatography (HPLC, RP 18).

JF 43 Maleimide

100 mg of JF 43 (0.16 mmol) are dissolved in 10 ml of dried DMSO andtreated with 100 mg (1 mmol) of maleic anhydride. The solution isstirred at room temperature for 24 h. 50 ml of 10% strength aqueoussodium perchlorate solution are added dropwise and the solidprecipitated is filtered off. The solid is suspended in 5 ml of aceticanhydride with 25 mg of sodium acetate and heated to 80° C. for 30 min.The mixture is cooled and 30 ml of 10% strength aqueous sodiumperchlorate solution are added dropwise. The solid is filtered off anddried.

JF 43-cysteine Conjugate

70 mg (0.1 mmol) of JF 43 maleimide are dissolved in 20 ml of ethanoland treated in portions with 12 mg (0.1 mmol) of cysteine. The solutionis stirred at room temperature for 30 min. After this, 50 ml of 10%strength aqueous sodium perchlorate solution are added dropwise and thesolid precipitated is filtered off and dried.

JA 262-dUTP Conjugate

10 μmol of 5-(3-aminoallyl)-dUTP are dissolved in 0.5 ml of 0.1 M sodiumborate buffer (pH 8) and treated with a solution of 5 μmol of JA 262active ester in 1 ml of amine-free dimethylformamide. The solution isstirred at room temperature for 15 h. The solvents are distilled off invacuo and the residue is purified by chromatography (RP 18).

JA 262-digoxin-3-carboxymethyl Ether-diaminodioxaoctane Conjugate(Dig-CME-DADOO)

0.02 mmol of JA 262 active ester are stirred in acetonitrile at roomtemperature for 18 h with 0.02 mmol of Dig-CME-DADOO. The solvent isdistilled off and the residue is purified by chromatography.

What is claimed is:
 1. In an immunoassay or nucleic acid hybridization method for the detection of an analyte in a sample, the improvement which comprises using a labeled receptor for the analyte wherein the label is a compound of the general formula I

wherein: R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are, independently, hydrogen, halogen, a hydroxyl, amino, sulfo, carboxyl or aldehyde group, a substituted or unsubstituted, saturated or unsaturated straight chain, branched or cyclic alkyl group having up to 20 carbon atoms, or a substituted or unsubstituted, aromatic ring system; or two or more adjacent R₁-R₇ groups, together may form a ring system which containing one or more multiple bonds; R₈ and R_(8a) are, independently, a saturated or unsaturated, straight-chain, branched or cyclic alkyl group having up to 20 carbon atoms, or R₈ and R_(8a), together, can form a ring system of one or more rings; R₉, R₁₀, R₁₁ and R₁₂, are independently, hydrogen, a substituted or unsubstituted, saturated or unsaturated, straight-chain, branched or cyclic alkyl group having up to 20 carbon atoms, a polyether, substituted or unsubstituted phenyl or substituted or unsubstituted phenylalkyl having 1-3 carbon atoms in the alkyl chain, provided that any of these groups may optionally contain one or more atoms selected from the group consisting of oxygen, sulfur and nitrogen atoms; or two or more adjacent R₉-R₁₂ groups, together, may form a ring system which can contain one or more multiple bonds; with the proviso that if R₁-R₃ and R₅-R₇ are hydrogen and R₈, R_(8a) and R₉-R₁₂ are methyl, then R₄ is not one of hydrogen, hydroxyl, methyl, isopropyl, t-butyl, phenyl, o-tolyl, p-tolyl, 2,6-dimethylphenyl, 2-t-butylphenyl, 2-isopropenylphenyl and 4-dimethylaminophenyl; wherein either or both of the groups —N(R₁₁)(R₁₂) and ═N(R₉)(R₁₀) can be replaced by either —OR₉ or ═O; and X represents a species of anion present for charge equalization.
 2. The method according to claim 1, wherein R₁-R₇ are, optionally, substituted by a member selected from the group consisting of halogens, hydroxyl, amino, sulfo, phospho, carboxyl, aldehyde, C₁-C₄-alkoxy, and C₁-C₄-alkoxycarbonyl groups.
 3. The method according to claim 1, wherein R₈-R_(8a) are, optionally, substituted with a member selected from the group consisting of halogens, hydroxyl, amino, sulfo, phospho, carboxyl, aldehyde, C₁-C₄-alkoxy and C₁-C₄-alkoxycarbonyl groups.
 4. The method according to claim 1, wherein R₉, R₁₀, R₁₁ and R₁₂ are, optionally, substituted with a member selected from the group consisting of halogens, hydroxyl, amino, sulfo, phospho, carboxyl, carbonyl, alkoxy and alkoxycarbonyl groups.
 5. The method as claimed in claim 1, wherein the compound I is covalently coupled to a receptor specific for an analyte to be detected.
 6. The method as claimed in claim 1, wherein the detection procedure is selected from nucleic acid hybridization procedures and immunochemical procedures.
 7. A compound of the general formula I

wherein R₁-R₁₂ and X are defined as in claim 1, with the proviso that if R₁-R₃ and R₅-R₇ are hydrogen and R₈, R_(8a) and R₉-R₁₂ are methyl, then R₄ is not one of hydrogen, hydroxyl, methyl, isopropyl, t-butyl, phenyl, o-tolyl, p-tolyl, 2,6-dimethylphenyl, 2-t-butylphenyl, 2-isopropenylphenyl and 4-dimethylaminophenyl, and wherein one or more ring systems are formed by R₆ bridging with R₁₁, R₇ bridging with R₁₂; R₁ bridging with R₉ and/or R₂ bridging with R₁₀.
 8. The compound according to claim 7, wherein the ring system formed by bridging R₆ with R₁₁, R₇ with R₁₂, R₁ with R₉ or R₂ with R₁₀ contains a 5- or 6-membered ring which contain one or more multiple bonds.
 9. The compound according to claim 7, wherein R₄ is hydrogen, C₁-C₆-alkyl or an aromatic ring system.
 10. The compound according to claim 7, wherein R₈ and R_(8a) are, independently, methyl, ethyl or phenyl.
 11. The compound according to claim 7, which corresponds to one of the general formulae IVa to IVe as follows:

in which the broken lines represent optional double bonds, and when the double bond is present in the ring the radicals R bonded via a broken line are absent; R₁, R₃, R₄, R₅, R₆, R₇, R_(8a), R₉, R₁₁, R₁₂ and X are as previously defined, and R in each occurrence, can be identical or different and is defined as R₁-R₇.
 12. The compound according to claim 7 further comprising a group capable of covalent coupling the compound to a biomolecule.
 13. The compound according to claim 12, wherein the coupling group is selected from the group consisting of —COOH, —NH2, —OH and —SH.
 14. The compound according to claim 12 which is coupled to at least one of a carrier and a biomolecule via one or more coupling groups.
 15. The compound according to claim 14, wherein the carrier is selected from the group consisting of porous glass, ion exchange resins, dextrans, cellulose, cellulose derivatives and hydrophilic polymers.
 16. The compound according to claim 14, wherein the biomolecule is selected from the group consisting of at peptides, polypeptides, nucleotides, nucleosides, nucleic acids, nucleic acid analogs and haptens.
 17. A process for the preparation of compounds of the general formula I

wherein R₁-R₁₂ and X are defined as in claim 1, comprising the steps of: reacting one of a compound of the general formula II

 in which R₅, R₆, R₇, R₈, R_(8a), R₁₁, R₁₂ are as previously defined, or the dehydration product of II, with a compound of the general formula III

 in which R₁-R₄, R₉ and R₁₀ are as previously defined and Y is a halogen, in a suitable solvent, under acidic conditions and in the presence of a catalyst; and reacting the compound formed by ring closure between one of the compound II or its dehydration product, and the compound III, by oxidation into the compound I.
 18. The process according to claim 17, wherein the solvent is a nonpolar solvent, selected from one of methylene chloride, 1,2-dichloroethane or chloroform.
 19. The process according to claim 17, wherein the catalyst is boron trichloride.
 20. The process according to claim 17, wherein the acid is selected from one of sulphuric acid, phosphoric acid or polyphosphoric acid.
 21. The process according to claim 17, wherein the oxidant is tetrabutylammonium(meta)periodate.
 22. The process according to claim 17, wherein the compound (I) is obtained in a one-step process and without isolation of intermediates.
 23. The method of claim 1, wherein the alkyl groups include at least one of phenyl and heteroaryl radicals as a substituent.
 24. The method of claim 23, wherein the aromatic ring system includes at least one heteroatom selected from oxygen, sulfur or nitrogen atoms and two or more substituents.
 25. The method of claim 1, wherein the saturated or unsaturated, straight-chain, branched or cyclic alkyl group having up to 20 carbon atoms is selected from the group consisting of at methyl, ethyl, propyl and butyl.
 26. The method of claim 1, wherein the aromatic ring system contains at least one of a heteratom selected from oxygen, sulfur or nitrogen atoms and one or more substituents.
 27. The process of claim 17, wherein the halogen is selected from the group of bromine, chorine or iodine.
 28. A conjugate for the detection of an analyte comprising a compound according to claim 7, which is covalently coupled to a receptor specific for an analyte to be detected. 